Project Summary/Abstract Apoptosis or programmed cell death is a fundamental and altruistic process that is critical for the removal of unneeded or aberrant cells during differentiation, viral infection or DNA damage. Induction of apoptosis is a complex signaling process involving many checks and balances that ultimately terminates with the activation of protein degrading enzymes known as caspases. The flow of this process proceeds in the following general sequence: intrinsic or extrinsic apoptotic signals->initiator caspases (-2, -8, or -9)->executioner caspases (-3, - 6 and/or -7)->cleavage of target proteins->cell death.The long-range goal of this proposal is to gain a fundamental understanding of the terminal process in apoptosis that is mediated by executioner caspases-3, - 6, and -7 and the targets they cleave in normal and cancer cells. Direct activation of inactive executioner procaspases to active caspases would allow one to bypass complex and lengthy signaling pathways so that executioner effects can be ascribed more clearly, and independent of upstream initiator caspases. Many cancer drugs work by inducing apoptosis indirectly through DNA damage or by inhibition of oncogenic proliferation pathways. Direct procaspase activator compounds would not induce DNA damage, nor be readily resisted by upstream signaling mutations. We also aim to develop a small molecule activated site-specific protease so that each caspase can be separately activated orthogonally and the cellular consequences tested. These studies will be supported by the global analysis of hundreds of protein substrates cleaved as a function of time using a novel proteomic approach (degradomics). These studies will greatly clarify the mechanisms and roles of executioner caspases, establish their validity as drug targets, and provide potential lead compounds for treating cancer. This work is broken into three specific aims which are supported by a wealth of preliminary data: Specific Aim 1. Identification and characterization of small molecules that activate executioner caspases. Specific Aim 2. Generation of an orthogonal means to selectively activate executioner procaspases in cells using a small molecule-activated TEV protease, and characterization of caspase-cleaved proteins by global proteomic analysis of the apoptotic program. Specific Aim 3. Determine the ability of broad and isoform-specific small molecule activators of executioner caspases to induce apoptosis in a range of cancer cells, non-transformed cells, and in engineered HEK-293 cells where we vary apoptotic components. These studies will yield potential lead compounds for inducing apoptosis and an understanding of their therapeutic window for inducing cell death in cancer versus normal cells. In addition, new technologies and chemical tools will be developed to aid in modulating and elucidating apoptosis. Project Narrative The proposed studies will result in the discovery of potential anti-cancer drug leads for broad or selective procaspase activation, that are mechanistically non-mutagenic and would be difficult to develop resistance to by typical oncogenic upstream mutations. Moreover, we will define the therapeutic window for these compounds in cells and potential pharmacodynamic and biomarkers for transitioning these to animal studies and ultimately humans.